The subject of the present invention is a new genetic material encoding a new protein and its fragments and its antigenic determinant recognized by anti-Trypanosoma cruzi antisera, and it relates to promoter sequence and to the use of said gene and protein and/or antigenic determinant, especially for diagnostic, pharmaceutical and therapeutic purposes.
Trypanosoma cruzi is a flagellate protozoan parasite, a member of the order Kinetoplastida and of the family Trypanosomatidae, which is responsible for Chagas"" disease which affects naturally millions of persons, mainly in Latin America.
In vertebrate hosts, Trypanosoma cruzi is present in two forms: one which is mobile by means of its flagellum or trypomastigote and which does not divide; the other is aflagellate, or intracellular amastigote, which multiplies by binary division.
Transmission of the protozoan in man occurs through hematophagous insects of the family Reduviidae, during a blood meal followed by dejections at the site of the bite. The vector insect thus releases the infectious metacyclic trypomastigote forms which will colonize many cell types through the blood circulation. Trypanosoma cruzi infects cardiac and skeletal muscle cells, glial cells, and cells of the mononuclear phagocytic system. After passive penetration into the host cell, the trypomastigote form of the parasite differentiates into the amastigote form, divides actively and then this is followed by a release of the trypomastigote forms, thereby causing a new cell invasion.
The vector insects will complete the parasitic cycle by ingesting, during a blood meal, the trypomastigote forms in the host. The latter differentiate into epimastigote forms in the vector""s middle intestine and finally into the infectious metacyclic trypomastigote forms in the posterior intestine.
Two phases can be distinguished in the Chagas disease: the acute phase and the chronic phase. The acute phase occurs after a transfusional, congenital, or vectorial type contamination and lasts for a few weeks. It is characterized by a large number of parasites circulating in the blood and corresponds to an exponential division of the protozoan. The acute phase is most often asymptomatic. However, in infants contaminated by their mother, the acute phase, which is marked by an acute cardiopathy, may be critical. The chronic phase may extend over many years. In some individuals, this phase is asymptomatic. On the other hand, other patients have tissue lesions in the heart or digestive type manifestations. In any case, clinical diagnosis must always be confirmed by tests for the detection either of antibodies directed against the parasitic antigens, or of the parasite itself.
This disease is becoming a worldwide problem because of the contamination through blood transfusion. It has therefore become essential to have available diagnostic tests which make it possible to determine the presence of the parasite in individuals. Various serological tests are avalable, such as direct agglutination, indirect immunofluorescence (IIF), complement fixation tests (CFR), and ELISA tests (Enzyme Linked Immunosorbent Assay). The Trypanosoma cruzi antigens currently used for the serological tests are obtained from a total lysate or from partially purified protein fractions of the noninfectious stage of the parasite. However, these fractions do not allow antigens to be obtained in sufficient quantity and quality for the production of a reliable serological diagnostic test. Furthermore, the complexity of the parasite and the strain-to-strain antigenic polymorphism introduce an additional difficulty in the reproducibility of the different preparations. Finally, there are many risks of cross-reactivity with other protozoa, more particularly with the family Leishmania and Trypanosoma rangeli, a nonpathogenic parasite.
In order to solve these various problems, it was envisaged to produce a serological diagnostic kit composed of recombinant proteins which would be highly sensitive and specific for Trypanosoma cruzi. 
Various research groups have screened libraries for expression of Trypanosoma cruzi genomic DNA or complementary DNA in the vector xcexgt11, using sera from patients suffering from Chagas disease. The xcexgt11 phage allows the insertion of foreign DNA of a maximum size of 7 kb into the EcoRI site localized in the lacZ gene, under the control of the lac promoter. The product obtained is a recombinant protein fused with beta-galactosidase, which is inducible by IPTG (isopropyl beta-D-thiogalactoside).
Various Trypanosoma cruzi genes, encoding proteins recognized by the Chagasic sera were thus characterized (Moncayo and Luquetti, 1990 and Levin et al. (1991), FEMS Microbiol. Immunol. 89: 11-20). Among the recombinant antigens described, the H49 antigen may be mentioned (Paranhos et al., 1994 (1)). However, this antigen does not allow a serological detection sensitivity of 100% of the patients in the acute or chronic phase. It was therefore envisaged to combine the H49 antigen with the CRA antigen (Cytoplasmic Repetitive Antigen) (Lafaille et al., (1989) (2)) but still without solving this problem.
The present inventors have identified and obtained a new genetic material encoding a new protein, its fragments, and antigenic determinants recognized by anti-Trypanosoma cruzi antisera, which makes it possible to overcome the above-mentioned disadvantages. The genetic material may be used to produce proteins or polypeptides for the production of diagnostic tests, or for the preparation of vaccinal or pharmaceutical compositions, or may itself either be used as a probe, or for the determination of specific probes which can be used in nucleic acid hybridization tests for the detection of Trypanosoma cruzi infections. Likewise, the protein or any corresponding polypeptide may be used for the production of antibodies specific for the parasite, for diagnostic or passive protection purposes.
The new genetic material is designated Tc100 and it encodes a protein designated PTc100 by the applicant. The new genetic material and protein have also been designated Tc40 and PTc40, respectively, by the applicant.
Consequently, the subject of the present invention is a DNA or RNA molecule consisting of at least one strand comprising a nucleotide sequence represented by the identifier SEQ ID No.1, or a sequence complementary or antisense or equivalent to said sequence identified by the identifier SEQ ID No.1, and especially a sequence having, for any succession of 100 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at least 85% homology with said sequence.
The invention moreover relates to DNA or RNA fragments whose nucleotide sequence is identical, complementary, antisense, or equivalent to any one of the following sequences:
that starting at nucleotide 1232 and ending at nucleotide 2207 of SEQ ID No.1,
that starting at nucleotide 1232 and ending at nucleotide 1825 of SEQ ID No.1,
and that starting at nucleotide 1266 and ending at nucleotide 2207, and especially the DNA or RNA fragments whose sequence has, for any succession of 30 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at least 85% homology with any one of said sequences.
The prefered DNA and RNA fragment being the 1232-1825 fragment of SEQ ID No.1, the invention relates to DNA or RNA fragments which hybridize with this fragment knowing that it is tolerated until 10% of error in bases"" matching.
The DNA and RNA fragment could be of any length and be comprised in the sequence from 266 to 3010 of SEQ ID No.1, knowing that this sequence is highly specific for Trypanosoma cruzi. Any fragment which hybridizes with the DNA or RNA of this sequence is considered as been a specific equivalent nucleotide or deoxunucleotide sequence.
Nucleotide sequence is understood to mean either a DNA strand or its complementary strand, or an RNA strand or its antisense strand or their corresponding complementary DNAs. The DNA sequence as represented in the identifier SEQ ID No.1 corresponds to the messenger RNA sequence, it being understood that the thymine (T) in the DNA is replaced by a uracil (U) in the RNA.
A still more prefered DNA and RNA fragment is the sequence from nucleotides 1472 to 1543 of SEQ ID No.1.
According to the invention, two nucleotide sequences are said to be equivalent in relation to each other, or in relation to a reference sequence if, functionally, the corresponding biopolymers can play essentially the same role, without being identical, with respect to the application or use considered, or in the technique in which they are involved; two sequences obtained because of the natural variability, especially spontaneous mutation, of the species from which they were identified, or because of induced variability, as well as homologous sequences, homology being defined below, are especially equivalent.
Variability is understood to mean any spontaneous or induced modification of a sequence, especially by substitution and/or insertion and/or deletion of nucleotides and/or of nucleotide fragments, and/or extension and/or shortening of the sequence at at least one of the ends; a nonnatural variability may result from the genetic engineering techniques used; this variability may result in modifications of any starting sequence, considered as reference, and capable of being expressed by a degree of homology relative to the said reference sequence.
Homology characterizes the degree of identity of two nucleotide (or peptide) fragments compared; it is measured by the percentage of identity which is especially determined by direct comparison of nucleotide (or peptide) sequences, relative to reference nucleotide (or peptide) sequences.
Any nucleotide fragment is said to be equivalent to a reference fragment if it has a nucleotide sequence which is equivalent to the reference sequence; according to the preceding definition, the following are especially equivalent to a reference nucleotide fragment:
a) any fragment capable of at least partially hybridizing with the complementary strand of the reference fragment, knowing that it is tolerated until 10% of error in bases"" matching.
b) any fragment whose alignment with the reference fragment leads to the detection of identical contiguous bases, in greater number than with any other fragment obtained from another taxonomic group,
c) any fragment resulting or capable of resulting from the natural variability of the species, from which it is obtained,
d) any fragment capable of resulting from the genetic engineering techniques applied to the reference fragment,
e) any fragment, containing at least 30 contiguous nucleotides, encoding a peptide homologous or identical to the peptide encoded by the reference fragment, and/or
f) any fragment different from the reference fragment by insertion, deletion, or substitution of at least one monomer, extension or shortening at at least one of its ends; for example any fragment corresponding to the reference fragment flanked at at least one of its ends by a nucleotide sequence not encoding a polypeptide.
The subject of the invention is also a protein, called PTc100 by the applicant, having an apparent molecular mass of about 100 kDa, which is recognized by anti-Trypanosoma cruzi antisera, or an immunological equivalent of this protein, and fragments thereof. The amino acid sequence of this protein is represented by the identifier sequence SEQ ID No.2, the sequence starting at amino acid 1 and ending at amino acid 915 of the sequence defined in the identifier SEQ ID No:2.
Immunological equivalent is understood to mean any polypeptide or peptide capable of being immunologically recognized by antibodies directed against a particular protein fragment.
The invention also relates specifically to a fragment of this protein represented by the identifier sequence SEQ ID No.2, the sequence starting at amino acid 323 and ending at amino acid 520 of the sequence defined in the identifier SEQ ID No.2.
The invention also relates specifically to the antigenic determinant or epitope of the PTc100 protein, a fragment of 25 amino acids, starting at amino acid 402 and ending at amino acid 426 of SEQ ID No.2, named S23G.
The PTc100 protein and said PTc100 fragment, as well as the S23G antigenic determinant may contain modifications, especially chemical modifications, which do not alter their antigenicity, such as for example N-terminal linkage by oxidation of serine or C-terminal linkage by cysteine function or hydrazine (see Keith Rose and al., Natural Peptides as Building blocks for the synthesis of large Protein-like Molecules with Hydrazone and Oxime Linkages. Department of Medical Biochemistry, (1996)).
PTc100 protein fragment is understood to mean any fragment of the PTc100 protein which preferably has at least the sequence from amino acid 323 to 520 of SEQ ID No. 2 and/or the S23G sequence, together with some other amino acids. It is clear that the S23G fragment contains an antigenic epitope of the PTc100 protein that reacts with antibodies generated during Chagas"" disease.
Moreover, the subject of the present invention is also an expression cassette which is functional especially in a cell derived from a prokaryotic or eukaryotic organism, and which allows the expression of DNA encoding the entire PTc100 protein or a fragment thereof, in particular of a DNA fragment as defined above, placed under the control of elements necessary for its expression; said protein and said protein fragments being recognized by anti-Trypanosoma cruzi antisera.
Generally, any cell derived from a prokaryotic or eukaryotic organism can be used within the framework of the present invention. Such cells are known to persons skilled in the art. By way of examples, there may be mentioned cells derived from a eukaryotic organism, such as the cells derived from a mammal, especially CHO (Chinese Hamster Ovarian) cells; insect cells; cells derived from a fungus, especially a unicellular fungus or from a yeast, especially of the strain Pichia, Saccharomyces, Schizosaccharomyces and most particularly selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae, Schizosaccharomyces octosporus, or other systems such as Semliki forest virus and vaccinia virus. Likewise, among the cells derived from a prokaryotic organism, there may be used, without this constituting a limitation, the cells of a strain of Escherichia coli (E. coli) or other enterobacterial cells. A large number of these cells are commercially available in collections, such as ATCC (Rockville, Md., USA) and AFRC (Agriculture and Food Research Council, Norfolk, UK). The cell may also be of the wild-type or mutant type. The mutations are described in the literature accessible to persons skilled in the art.
For the purposes of the present invention, an E. coli DH5xcex1 cell (marketed by the company CLONTECH under the reference: C2007-1) is used; however, other cells may be used as well.
The expression cassette of the invention is intended for the production of the PTc100 protein or for fragments of said protein, such as the PTc100 fragment and/or the S23G epitope, which are produced by the above mentioned E. coli cell, and which are recognized by human antisera. Such antisera are obtained from patients who have contracted a Trypanosoma cruzi infection recently or long ago, and contain immunoglobulins specifically recognizing the PTc100 protein and/or a PTc100 protein fragment. Of course, the PTc100 protein can also be recognized by other antibodies, such as for example monoclonal or polyclonal antibodies obtained by immunization of various species with the natural above-mentioned protein, the recombinant protein, or fragments or peptides thereof.
PTc100 protein is understood to mean the native Trypanosoma cruzi cytoplasmic antigen, or the antigen produced especially by the genetic recombination techniques described in the present application, or any fragment or mutant of this antigen, for exemple the GST-Tc100 recombinant protein and the BIO-S23G (Tc40) peptide, provided that it is immunologically reactive with antibodies directed against the PTc100 protein of this parasite.
Advantageously, such a protein has an amino acid sequence having a degree of homology of at least 70%, preferably of at least 85%, and most preferably of at least 95% relative to the sequence identified by the identifier SEQ ID No.2. In practice, such an equivalent can be obtained by deletion, substitution, and/or addition of one or more amino acids of the recombinant protein sequence. Such homology means that the amino acid sequence of the S23G peptide, contained in the PTc100 protein and its PTc100 protein fragment, is at least preserved in order to keep the antigenicity potentiality. It is within the capability of persons skilled in the art to perform, using known techniques, these modifications without affecting immunological recognition.
Within the framework of the present invention, the PTc100 protein and the PTc100 protein fragment can be modified in vitro, especially by deletion or addition of chemical groups, such as phosphates, sugars or myristic acids, so as to enhance its antigenic stability or the presentation of one or several epitopes, such as the S23G epitope.
The expression cassette according to the invention allows the production of a PTc100 protein (having an amino acid sequence as specified above) and fragments of said protein, fused with an exogenous element which can help its stability, its purification, its production, its presentation, or its recognition. The choice of such an exogenous element is within the capability of persons skilled in the art. It may be especially a hapten, an exogenous peptide, or a protein.
The expression cassette according to the invention comprises the elements necessary for the expression of said DNA fragment in the cell considered. xe2x80x9cElements necessary for the expressionxe2x80x9d is understood to mean the elements as a whole which allow the transcription of the DNA fragment into messenger RNA (mRNA) and the translation of the latter into protein.
The present invention also relates to a putative promoter, specific for T. cruzi, which can be used in an expression cassette for eukaryotic expression of genes. Probes could also be defined in this region to detect the parasite.
The present invention also extends to a vector comprising an expression cassette according to the invention. This may be a viral vector and especially a vector derived from a baculovirus, more particularly intended for expression in insect cells, or an adenovirus-derived vector for expression in mammalian cells. It may also be an autonomously replicating plasmid vector and in particular a multiplicative vector.
The present invention also relates to a cell derived from a prokaryotic or eukaryotic organism, comprising an expression cassette, either in a form integrated in the cellular genome, or inserted in a vector which can be maintained as an extra-genomic entity. Such a cell was previously defined.
The subject of the present invention is also a process for preparing a PTc100 protein, or fragments of said protein, according to which:
(i) a cell derived from a prokaryotic or eukaryotic organism, comprising the expression cassette according to the invention, is cultured under appropriate conditions; and
(ii) the expressed protein derived from the above mentioned organism is recovered.
The present invention also relates to one or more peptides, whose amino acid sequence corresponds to a portion of the sequence of the PTc100 protein and exhibits, alone or as a mixture, a reactivity with the entire sera from individuals or animals infected with Trypanosoma cruzi. The prefered portion is the PTc100 protein fragment or peptide defined above and the S23G peptide. The peptides can be obtained by chemical synthesis, lysis of the PTc100 protein, or by genetic recombination techniques.
The invention also relates to monoclonal or polyclonal antibodies obtained by immunological reaction of a human or animal organism to an immunogenic agent consisting of the natural or recombinant PTc100 protein and fragments thereof, or of a peptide, as defined above.
The present invention also relates to a reagent for the detection and/or monitoring of a Trypanosoma cruzi infection, which comprises, as reactive substance, a PTc100 protein as defined above, or fragments thereof, a peptide or a mixture of peptides as defined above, or at least one monoclonal or polyclonal antibody as described above. The above reagent may be attached directly or indirectly to an appropriate solid support. The solid support may be especially in the form of a cone, a tube, a well, a bead, and the like.
The term xe2x80x9csolid supportxe2x80x9d as used here includes all materials on which a reagent can be immobilized for use in diagnostic tests. Natural or synthetic materials, chemically modified or otherwise, can be used as solid supports, especially polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose; polymers such as vinyl chloride, polyethylene, polystyrenes, polyacrylate or copolymers such as polymers of vinyl chloride and propylene, polymers of vinyl chloride and vinyl acetate; styrene-based copolymers, natural fibers such as cotton and synthetic fibers such as nylon; and magnetic particles. Preferably, the solid support is a polystyrene polymer or a butadiene/styrene copolymer. Advantageously, the support is a polystyrene or a styrene-based copolymer comprising between about 10 and 90% by weight of styrene units.
The binding of the reagent onto the solid support may be performed in a direct or indirect manner. Using the direct manner, two approaches are possible: either by adsorption of the reagent onto the solid support, that is to say by noncovalent bonds (principally of the hydrogen, Van der Waals or ionic type), or by formation of covalent bonds between the reagent and the support. Using the indirect manner, an xe2x80x9canti-reagentxe2x80x9d compound capable of interacting with the reagent so as to immobilize the whole onto the solid support can be attached beforehand (by adsorption or covalent bonding) onto the solid support. By way of example, there may be mentioned an anti-PTc100 antibody, on the condition that it is immunologically reactive with a portion of the protein different from that involved in the reaction for recognizing the antibodies in the sera; a ligand-receptor system, for example by grafting onto the PTc100 protein a molecule such as a vitamin, and by immobilizing onto the solid phase the corresponding receptor (for example the biotin-streptavidin system). Indirect manner is also understood to mean the preliminary grafting or fusion by genetic recombination of a protein, or a fragment of this protein, or of a polypeptide, to one end of the PTc100, protein, and the immobilization of the latter onto the solid support by passive adsorption or covalent bonding of the protein or of the polypeptide grafted or fused.
The invention also relates to a process for the detection and/or monitoring of a Trypanosoma cruzi infection in a biological sample, such as blood serum or plasma, urine, saliva, or tear samples from an individual or an animal likely to have been infected with Trypanosoma cruzi, characterized in that said sample and a reagent as defined above are placed in contact, under conditions allowing a possible immunological reaction, and the presence of an immune complex with said reagent is then detected.
By way of non-limiting example, there may be mentioned the sandwich-type detection process in one or more stages, as especially described in patents FR 2,481,318 and FR 2,487,983, which consists of reacting a first monoclonal or polyclonal antibody specific for a desired antigen, attached onto a solid support, with the sample, and in revealing the possible presence of an immune complex thus formed using a second antibody labelled by any appropriate marker known to persons skilled in the art, especially a radioactive isotope, an enzyme, for example peroxidase or alkaline phosphatase and the like, using so-called competition techniques well known to persons skilled in the art.
The subject of the invention is also an active immunotherapeutic composition, especially a vaccine preparation, which comprises as active ingredient, a natural or recombinant PTc100 protein or fragments thereof, or the peptides identified above, the active ingredient being optionally conjugated with a pharmaceutically acceptable carrier, and optionally an excipient and/or an appropriate adjuvant.
The present invention also covers a pharmaceutical composition intended for the treatment or for the prevention of a Trypanosoma cruzi infection in man or in an animal, comprising a therapeutically effective quantity of an expression cassette, a vector, a cell derived from a prokaryotic or eukaryotic organism as defined above, a PTc100 protein according to the invention, or fragments thereof, or the peptide identified above, or an antibody of the invention.
The subject of the present invention is also probes and primers specific for T. cruzi, and their uses in diagnostic tests.
The term probe as used in the present invention refers to a DNA or RNA containing at least one strand having a nucleotide sequence which allows hybridization to nucleic acids having a nucleotide sequence as represented by the identifier SEQ ID No.1, or a complementary or antisense sequence, or a sequence equivalent to said sequence, and especially a sequence having, for any succession of 5 to 100 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at least 85% homology with SEQ ID No.1, with fragments thereof, or with a synthetic oligonucleotide allowing such a hybridization, nonmodified or comprising one or more modified bases such as inosine, 5-methyldeoxycytidine, deoxyuridine, 5-dimethylaminodeoxyuridine, 2,6-diaminopurine, 5-bromodeoxyuridine or any other modified base. Likewise, these probes may be modified at the level of the sugar, namely the replacement of at least one deoxyribose with a polyamide (P. E. NIELSEN et al. (1991) (13)), or at the level of the phosphate group, for example its replacement with esters, especially chosen from esters of diphosphate, of alkyl and arylphosphonate and of phosphorothioate.
The probes may be much shorter than the sequence identified in the identifier SEQ ID No.1. In practice, such probes comprise at least 5 monomers, advantageously from 8 to 50 monomers, having a hybridization specificity, under defined conditions, to form a hybridization complex with DNA or RNA having a nucleotide sequence as defined above. The conditions are well-known and/or easily determined by those of skill in the art.
A probe according to the invention can be used for diagnostic purposes, such as primer for amplification of biological material, as capture and/or detection probe, or for therapeutic purposes. The capture probe can be immobilized on a solid support by any appropriate means, that is to say directly or indirectly, for example by covalent bonding or passive adsorption. The detection probe is labelled by means of a marker chosen from radioactive isotopes, enzymes especially chosen from peroxidase and alkaline phosphatase, and those capable of hydrolyzing a chromogenic, fluorigenic, or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorigenic, or luminescent compounds, nucleotide base analogs, and biotin.
The probes of the present invention which are used for diagnostic purposes can be used in any known hybridization techniques, and especially the so-called xe2x80x9cDot-Blotxe2x80x9d technique (Maniatis et al. (1982) (14)), Southern Blotting technique (Southern E. M. (1975) (15)), Northern Blotting technique, which is a technique identical to the Southern Blotting technique but which uses RNA as target, sandwich technique (Dunn A. R. et al. (1977) (16)). Advantageously, the sandwich tech-nique is used which comprises a specific capture probe and/or a specific detection probe, it being understood that the capture probe and the detection probe must have a nucleotide sequence which is at least partially different.
Another application of the invention is a therapeutic probe for treating infections due to Trypanosoma cruzi, said probe being capable of hybridizing in vivo with the DNA or RNA of the parasite to block the translation and/or transcription and/or replication phenomena.
A primer is a probe comprising 5 to 30 monomers, having a hybridization specificity, under predefined conditions, for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (Polymerase Chain Reaction), in an elongation process such as sequencing, in a reverse transcription method and the like. Such predefined conditions are well-known and/or easily determined by those of skill in the art.
A preferred probe or primer will contain a nucleotide sequence chosen from the sequences SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7.
The invention also relates to a reagent for detecting and/or identifying Trypanosoma cruzi in a biological sample, comprising at least one probe as defined above, and in particular a capture probe and a detection probe, either or both corresponding to the above definition.
The invention therefore provides a process for selectively detecting and/or for identifying Trypanosoma cruzi in a biological sample, according to which the RNA, extracted from the parasite and optionally denatured, or the DNA, denatured extract, or the DNA obtained from reverse transcription of the RNA, is exposed to at least one probe as defined above and the hybridization of said probe is detected.